title: Measuring protein interactions using Förster resonance energy transfer and fluorescence lifetime imaging microscopy.
abstract: The method of fluorescence lifetime imaging microscopy (FLIM) is a quantitative approach that can be used to detect Förster resonance energy transfer (FRET). The use of FLIM to measure the FRET that results from the interactions between proteins labeled with fluorescent proteins (FPs) inside living cells provides a non-invasive method for mapping interactomes. Here, the use of the phasor plot method to analyze frequency domain (FD) FLIM measurements is described, and measurements obtained from cells producing the 'FRET standard' fusion proteins are used to validate the FLIM system for FRET measurements. The FLIM FRET approach is then used to measure both homologous and heterologous protein-protein interactions (PPI) involving the CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a transcription factor that controls cell differentiation, and localizes to heterochromatin where it interacts with the heterochromatin protein 1 alpha (HP1α). The FLIM-FRET method is used to quantify the homologous interactions between the FP-labeled basic leucine zipper (BZip) domain of C/EBPα. Then the heterologous interactions between the C/EBPa BZip domain and HP1a are quantified using the FRET-FLIM method. The results demonstrate that the basic region and leucine zipper (BZip) domain of C/EBPα is sufficient for the interaction with HP1α in regions of heterochromatin.
authors.affiliation: Department of Cellular and Integrative Physiology, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202, USA. Electronic address: rnday@iupui.edu.
authors.forename: Richard N
authors.initials: RN
authors.lastname: Day
mesh_terms: D000818:Animals; D000848:Anodontia; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism; D004735:Energy Transfer; D031541:Fluorescence Resonance Energy Transfer / Q000592:standards; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D007180:Incisor / Q000002:abnormalities; D051379:Mice; D008856:Microscopy, Fluorescence / Q000592:standards; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012996:Solutions
chemical_list: C000715594:CBX5 protein, human; D022762:CCAAT-Enhancer-Binding Proteins; C515848:CEBPA protein, human; D002868:Chromosomal Proteins, Non-Histone; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D012996:Solutions; D000090266:Chromobox Protein Homolog 5; D049452:Green Fluorescent Proteins
keywords: Fluorescence lifetime imaging microscopy (FLIM); Fluorescent proteins (FPs); Förster resonance energy transfer (FRET) microscopy; Protein–protein interactions (PPI)